Human kinase and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application is a continuation of U.S. application Ser. No.10/014,882 filed Dec. 11, 2001 now U.S. Pat. No. 6,593,126, issued Jul.15, 2003, which claims the benefit of U.S. Provisional application No.60/254,744 which was filed on Dec. 11, 2000, each of which areincorporated by reference in their entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding a protein thatshares sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

Kinases mediate phosphorylation of a wide variety of proteins andcompounds in the cell. Along with phosphatases, kinases are involved ina range of regulatory pathways. Given their physiological importance,kinases have been subject to intense scrutiny and are proven drugtargets.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode a novel human protein, andthe corresponding amino acid sequence of this protein. The novel humanprotein (NHP) described for the first time herein shares structuralsimilarity with animal kinases, including, but not limited to,serine/threonine kinases, tyrosine kinases, TGF-beta activated kinases,and a variety of growth factor receptors. As such, the novelpolynucleotides encode a new kinase protein having homologues andorthologs across a range of phyla and species.

The novel human polynucleotides described herein, encode an open readingframe (ORF) encoding a protein of 1036 amino acids in length (see SEQ IDNO: 2).

The invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-3 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-3 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses.

Additionally, the unique NHP sequences described in SEQ ID NOS:1-3 areuseful for the identification of protein coding sequence and mapping aunique gene to a particular chromosome (the described NHP is apparentlyencoded on human chromosome 1, see GENBANK accession no. AL133380).These sequences identify actual, biologically verified, and thereforerelevant, exon splice junctions as opposed to those that may have beenbioinformatically predicted from genomic sequence alone. The sequencesof the present invention are also useful as additional DNA markers forrestriction fragment length polymorphism (RFLP) analysis, and inforensic biology.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists, of NHPexpression and/or NHP activity that utilize purified preparations of thedescribed NHPs and/or NHP product, or cells expressing the same. Suchcompounds can be used as therapeutic agents for the treatment of any ofa wide variety of symptoms associated with biological disorders orimbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequence of a novel human ORF thatencodes the described novel human kinase protein. SEQ ID NO:3 describesthe NHP ORF and flanking regions.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHP described for the first time herein, is a novel protein that isexpressed in, inter alia, human cell lines, and human fetal brain,brain, pituitary, cerebellum, lymph node, trachea, kidney, liver,prostate, testis, thyroid, adrenal gland, pancreas, stomach, smallintestine, colon, skeletal muscle, heart, uterus, and fetal kidneycells. The described sequences were compiled from human genomic sequenceand cDNAs made from human brain, lymph node, liver, cerebellum, kidney,testis, and bone marrow mRNAs (Edge Biosystems, Gaithersburg, Md.,Clontech, Palo Alto, Calif.).

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described genes, including the specifically describedNHP, and the NHP products; (b) nucleotides that encode one or moreportions of the NHP that correspond to functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the novel regions of any active domain(s); (c)isolated nucleotides that encode mutant versions, engineered ornaturally occurring, of the described NHP in which all or a part of atleast one domain is deleted or altered, and the polypeptide productsspecified by such nucleotide sequences, including but not limited tosoluble proteins and peptides in which all or a portion of the signalsequence is deleted; (d) nucleotides that encode chimeric fusionproteins containing all or a portion of a coding region of a NHP, or oneof its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing. As discussed above,the present invention includes: (a) the human DNA sequences presented inthe Sequence Listing (and vectors comprising the same) and additionallycontemplates any nucleotide sequence encoding a contiguous NHP openreading frame (ORF) that hybridizes to a complement of a DNA sequencepresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1%SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3) and encodes a functionallyequivalent expression product. Additionally contemplated are anynucleotide sequences that hybridize to the complement of the DNAsequence that encode and express an amino acid sequence presented in theSequence Listing under moderately stringent conditions, e.g., washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet stillencode a functionally equivalent NHP product. Functional equivalents ofa NHP include naturally occurring NHPs present in other species andmutant NHPs whether naturally occurring or engineered (by site directedmutagenesis, gene shuffling, directed evolution as described in, forexample, U.S. Pat. No. 5,837,458 or 5,723,323 both of which are hereinincorporated by reference). The invention also includes degeneratenucleic acid variants of the disclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHP ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package usingdefault parameters).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP encoding polynucleotides. Such hybridization conditionscan be highly stringent or less highly stringent, as described above. Ininstances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80, or about 34 to about 45 bases long, orany variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a micro array or high-throughput “chip” format).Additionally, a series of the described NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. An oligonucleotide or polynucleotidesequence first disclosed in at least a portion of one or more of thesequences of SEQ ID NOS: 1-3 can be used as a hybridization probe inconjunction with a solid support matrix/substrate (resins, beads,membranes, plastics, polymers, metal or metallized substrates,crystalline or polycrystalline substrates, etc.). Of particular note arespatially addressable arrays (i.e., gene chips, microtiter plates, etc.)of oligonucleotides and polynucleotides, or corresponding oligopeptidesand polypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the sequencesof SEQ ID NOS: 1-3, or an amino acid sequence encoded thereby. Methodsfor attaching biopolymers to, or synthesizing biopolymers on, solidsupport matrices, and conducting binding studies thereon are disclosedin, inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and 4,689,405 thedisclosures of which are herein incorporated by reference in theirentirety.

Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-3 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-3.

For example, a series of the described oligonucleotide sequences, or thecomplements thereof, can be used in chip format to represent all or aportion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

Microarray-based analysis allows the discovery of broad patterns ofgenetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-3 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

Probes consisting of sequences first disclosed in SEQ ID NOS:1-3 canalso be used in the identification, selection and validation of novelmolecular targets for drug discovery. The use of these unique sequencespermits the direct confirmation of drug targets and recognition of drugdependent changes in gene expression that are modulated through pathwaysdistinct from the drugs intended target. These unique sequencestherefore also have utility in defining and monitoring both drug actionand toxicity.

As an example of utility, the sequences first disclosed in SEQ IDNOS:1-3 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-3 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

Thus the sequences first disclosed in SEQ ID NOS:1-3 can be used toidentify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-3. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence. For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP gene antisense molecules, useful, for example,in NHP gene regulation and/or as antisense primers in amplificationreactions of NHP gene nucleic acid sequences. With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupincluding, but not limited to, a phosphorothioate, a phosphorodithioate,a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a form acetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Low stringency conditions are well-known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, NY.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, NY.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

For example, the present sequences can be used in restriction fragmentlength polymorphism (RFLP) analysis to identify specific individuals. Inthis technique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification (as generally described in U.S. Pat. No. 5,272,057,incorporated herein by reference). In addition, the sequences of thepresent invention can be used to provide polynucleotide reagents, e.g.,PCR primers, targeted to specific loci in the human genome, which canenhance the reliability of DNA-based forensic identifications by, forexample, providing another “identification marker” (i.e., another DNAsequence that is unique to a particular individual). Actual basesequence information can be used for identification as an accuratealternative to patterns formed by restriction enzyme generatedfragments.

Further, a NHP gene homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences within the NHP products disclosed herein. The template for thereaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue, such as prostate, rectum, colon, or adrenal gland,known or suspected to express an allele of a NHP gene.

The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known to, or suspectedof, expressing a NHP gene). A reverse transcription (RT) reaction can beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with acomplementary primer. Thus, cDNA sequences upstream of the amplifiedfragment can be isolated. For a review of cloning strategies that can beused, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP sequence can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown or suspected to be expressed in an individual putatively carryinga mutant NHP allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal sequence. Using these two primers, the product is then amplifiedvia PCR, optionally cloned into a suitable vector, and subjected to DNAsequence analysis through methods well-known to those of skill in theart. By comparing the DNA sequence of the mutant NHP allele to that of acorresponding normal NHP allele, the mutation(s) responsible for theloss or alteration of function of the mutant NHP gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant NHP allele(e.g., a person manifesting a NHP-associated phenotype such as, forexample, immune disorders, obesity, high blood pressure, etc.), or acDNA library can be constructed using RNA from a tissue known, orsuspected, to express a mutant NHP allele. A normal NHP gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHP allele in such libraries. Clonescontaining mutant NHP sequences can then be purified and subjected tosequence analysis according to methods well-known to those skilled inthe art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.)

Additionally, screening can be accomplished by screening with labeledNHP fusion proteins, such as, for example, alkaline phosphatase-NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expression product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toNHP are likely to cross-react with a corresponding mutant NHP expressionproduct. Library clones detected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well-known in the art.

An additional application of the described novel human polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721and 5,837,458 which are herein incorporated by reference in theirentirety.

The invention also encompasses (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

Where, as in the present instance, some of the described NHP peptides orpolypeptides are thought to be cytoplasmic proteins, expression systemscan be engineered that produce soluble derivatives of a NHP(corresponding to a NHP extracellular and/or intracellular domains, ortruncated polypeptides lacking one or more hydrophobic domains) and/orNHP fusion protein products (especially NHP-Ig fusion proteins, i.e.,fusions of a NHP domain to an IgFc), NHP antibodies, and anti-idiotypicantibodies (including Fab fragments) that can be used in therapeuticapplications. Preferably, the above expression systems are engineered toallow the desired peptide or polypeptide to be recovered from theculture media.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of a NHP,as well as compounds or nucleotide constructs that inhibit expression ofa NHP sequence (transcription factor inhibitors, antisense and ribozymemolecules, or open reading frame sequence or regulatory sequencereplacement constructs), or promote the expression of a NHP (e.g.,expression constructs in which NHP coding sequences are operativelyassociated with expression control elements such as promoters,promoter/enhancers, etc.).

The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences,antibodies, antagonists and agonists can be useful for the detection ofmutant NHPs or inappropriately expressed NHPs for the diagnosis ofdisease. The NHP proteins or peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHP inthe body. The use of engineered host cells and/or animals can offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor/ligand of a NHP, but canalso identify compounds that trigger NHP-mediated activities orpathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives such as NHP peptides/domains corresponding to theNHPs, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHP, or a NHP-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the NHP could activateor effectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, well as antisense and ribozyme molecules can also be usedin “gene therapy” approaches for the modulation of NHP expression. Thus,the invention also encompasses pharmaceutical formulations and methodsfor treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The NHP Sequences

The cDNA sequences and the corresponding deduced amino acid sequence ofthe described NHP are presented in the Sequence Listing. The NHPnucleotide sequences were obtained from cDNAs obtained using probesand/or primers generated from human genomic sequence.

A number of polymorphisms that may occur in the described NHP wereidentified including an A/G polymorphism at the nucleotide positionrepresented by, for example, position 2182 of SEQ ID NO: 1 (which canresult in a val or ile at the region corresponding to amino acid (aa)position 728 of, for example, SEQ ID NO:2), a G/T polymorphism atnucleotide position 2223 (which can result in an glu or asp at aaposition 741), a G/T polymorphism at nucleotide position 2350 (which canresult in a gly or cys at aa position 784), an A/C polymorphism atnucleotide position 2765 (which can result in an asp or ala at aaposition 922), a C/T polymorphism at nucleotide position 2768 (which canresult in a leu or pro at aa position 923), and an A/T polymorphism atnucleotide position 2773 (which can result in a ser or cys at aaposition 925).

Expression analysis has provided evidence that the described NHPs arepredominantly expressed in CNS tissues, and that the NHP sharessignificant similarity with a variety of protein kinases. Given thephysiological importance of protein kinases, they have been subject tointense scrutiny as exemplified and discussed in U.S. Pat. Nos.5,756,289 and 5,817,479 herein incorporated by reference in theirentirety which additionally describe a variety of uses and applicationsfor the described NHP.

The described NHP is apparently encoded on human chromosome 1.

The described novel human polynucleotide sequences can be used, amongother things, in the molecular mutagenesis/evolution of proteins thatare at least partially encoded by the described novel sequences using,for example, polynucleotide shuffling or related methodologies. Suchapproaches are described in U.S. Pat. Nos. 5,830,721 and 5,837,458 whichare herein incorporated by reference in their entirety.

NHP gene products can also be expressed in transgenic animals. Animalsof any species, including, but not limited to, worms, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, birds, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate NHP transgenic animals.

Any technique known in the art may be used to introduce a NHP transgeneinto animals to produce the founder lines of transgenic animals. Suchtechniques include, but are not limited to pronuclear microinjection(Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

The present invention provides for transgenic animals that carry the NHPtransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell-type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell-type ofinterest, and will be apparent to those of skill in the art.

When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

The transgene can also be selectively introduced into a particularcell-type, thus inactivating the endogenous NHP gene in only thatcell-type, by following, for example, the teaching of Gu et al., 1994,Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particularcell-type of interest, and will be apparent to those of skill in theart.

Once transgenic animals have been generated, the expression of therecombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

5.2 NHP and NHP Polypeptides

NHP products, polypeptides, peptide fragments, mutated, truncated, ordeleted forms of the NHPs, and/or NHP fusion proteins can be preparedfor a variety of uses. These uses include, but are not limited to, thegeneration of antibodies, as reagents in diagnostic assays, theidentification of other cellular gene products related to the NHP, asreagents in assays for screening for compounds that can be used aspharmaceutical reagents for the therapeutic treatment of mental,biological, or medical disorders and disease.

The Sequence Listing discloses the amino acid sequence encoded by thedescribed NHP-encoding polynucleotides. The NHP has an initiatormethionine in a DNA sequence context consistent with eucaryotictranslation initiation site and a signal-like sequence indicating thatthe NHP can be secreted or membrane associated.

The NHP amino acid sequence of the invention include the amino acidsequence presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto the NHP products encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHP substrate, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHPproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent gene product. Aminoacid substitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Where the NHP peptide orpolypeptide can exist, or has been engineered to exist, as a soluble orsecreted molecule, the soluble NHP peptide or polypeptide can berecovered from the culture media. Such expression systems also encompassengineered host cells that express a NHP, or functional equivalent, insitu. Purification or enrichment of a NHP from such expression systemscan be accomplished using appropriate detergents and lipid micelles andmethods well-known to those skilled in the art. However, such engineeredhost cells themselves may be used in situations where it is importantnot only to retain the structural and functional characteristics of theNHP, but to assess biological activity, e.g., in certain drug screeningassays.

The expression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign polynucleotide sequences.The virus grows in Spodoptera frugiperda cells. A NHP coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofNHP coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted sequence is expressed (e.g., see Smith et al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing a NHP product in infected hosts (e.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals may also be required for efficient translation of inserted NHPnucleotide sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire NHP gene or cDNA, includingits own initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of a NHPcoding sequence is inserted, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBitter et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe NHP sequences described above can be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the NHPproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the NHP product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺-nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

Also encompassed by the present invention are fusion proteins thatdirect the NHP to a target organ and/or facilitate transport across themembrane into the cytosol. Conjugation of NHPs to antibody molecules ortheir Fab fragments could be used to target cells bearing a particularepitope. Attaching the appropriate signal sequence to the NHP would alsotransport the NHP to the desired location within the cell. Alternativelytargeting of NHP or its nucleic acid sequence might be achieved usingliposome or lipid complex based delivery systems. Such technologies aredescribed in “Liposomes:A Practical Approach”, New, R.R.C., ed., OxfordUniversity Press, New York and in U.S. Pat. Nos. 4,594,595, 5,459,127,5,948,767 and 6,110,490 and their respective disclosures which areherein incorporated by reference in their entirety. Additionallyembodied are novel protein constructs engineered in such a way that theyfacilitate transport of the NHP to the target site or desired organ,where they cross the cell membrane and/or the nucleus where the NHP canexert its functional activity. This goal may be achieved by coupling ofthe NHP to a cytokine or other ligand that provides targetingspecificity, and/or to a protein transducing domain (see generally U.S.applications Ser. Nos. 60/111,701 and 60/056,713, both of which areherein incorporated by reference, for examples of such transducingsequences) to facilitate passage across cellular membranes and canoptionally be engineered to include nuclear localization.

5.3 Antibodies to NHP Products

Antibodies that specifically recognize one or more epitopes of a NHP, orepitopes of conserved variants of a NHP, or peptide fragments of a NHPare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the NHP, an NHP peptide (e.g., one corresponding to afunctional domain of an NHP), truncated NHP polypeptides (NHP in whichone or more domains have been deleted), functional equivalents of theNHP or mutated variant of the NHP. Such host animals may include but arenot limited to pigs, rabbits, mice, goats, and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including, but not limited to, Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, chitosan, surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, and potentially useful human adjuvants such as BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Alternatively, the immuneresponse could be enhanced by combination and or coupling with moleculessuch as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid,ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofthe immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal, Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused (see U.S. Pat. Nos. 6,075,181 and 5,877,397 both of which areherein incorporated by reference in their entirety). A chimeric antibodyis a molecule in which different portions are derived from differentanimal species, such as those having a variable region derived from amurine mAb and a human immunoglobulin constant region. Such technologiesare described in U.S. Pat. Nos. 6,075,181 and 5,877,397 and theirrespective disclosures which are herein incorporated by reference intheir entirety. Also encompassed by the present invention is the use offully humanized monoclonal antibodies as described in U.S. Pat. No.6,150,584 and respective disclosures which are herein incorporated byreference in their entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 341:544-546) can be adapted to produce single chainantibodies against NHP expression products. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: F(ab′)₂ fragments, which can be produced by pepsin digestionof the antibody molecule; and Fab fragments, which can be generated byreducing the disulfide bridges of the F(ab′)₂ fragments. Alternatively,Fab expression libraries may be constructed (Huse et al., 1989, Science,246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHP, using techniques well-known tothose skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). Forexample antibodies which bind to a NHP domain and competitively inhibitthe binding of NHP to its cognate receptor/ligand can be used togenerate anti-idiotypes that “mimic” the NHP and, therefore, bind,activate, or neutralize a NHP, NHP receptor, or NHP ligand. Suchanti-idiotypic antibodies or Fab fragments of such anti-idiotypes can beused in therapeutic regimens involving a NHP-mediated pathway.

Additionally given the high degree of relatedness of mammalian NHPs, thepresently described knock-out mice (having never seen NHP, and thusnever been tolerized to NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-outanimals).

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 3 <210> SEQ ID NO 1 <211> LENGTH: 3111<212> TYPE: DNA <213> ORGANISM: homo sapiens <400> SEQUENCE: 1atggctttgc ggggcgccgc gggagcgacc gacaccccgg tgtcctcggc cg#ggggagcc     60cccggcggct cagcgtcctc gtcgtccacc tcctcgggcg gctcggcctc gg#cgggcgcg    120gggctgtggg ccgcgctcta tgactacgag gctcgcggcg aggacgagct ga#gcctgcgg    180cgcggccagc tggtggaggt gctgtcgcag gacgccgccg tgtcgggcga cg#agggctgg    240tgggcaggcc aggtgcagcg gcgcctcggc atcttccccg ccaactacgt gg#ctccctgc    300cgcccggccg ccagccccgc gccgccgccc tcgcggccca gctccccggt ac#acgtcgcc    360ttcgagcggc tggagctgaa ggagctcatc ggcgctgggg gcttcgggca gg#tgtaccgc    420gccacctggc agggccagga ggtggccgtg aaggcggcgc gccaggaccc gg#agcaggac    480gcggcggcgg ctgccgagag cgtgcggcgc gaggctcggc tcttcgccat gc#tgcggcac    540cccaacatca tcgagctgcg cggcgtgtgc ctgcagcagc cgcacctctg cc#tggtgctg    600gagttcgccc gcggcggagc gctcaaccga gcgctggccg ctgccaacgc cg#ccccggac    660ccgcgcgcgc ccggcccccg ccgcgcgcgc cgcatccctc cgcacgtgct gg#tcaactgg    720gccgtgcaga tagcgcgggg catgctctac ctgcatgagg aggccttcgt gc#ccatcctg    780caccgggacc tcaagtccag caacattttg ctacttgaga agatagaaca tg#atgacatc    840tgcaataaaa ctttgaagat tacagatttt gggttggcga gggaatggca ca#ggaccacc    900aaaatgagca cagcaggcac ctatgcctgg atggcccccg aagtgatcaa gt#cttccttg    960ttttctaagg gaagcgacat ctggagctat ggagtgctgc tgtgggaact gc#tcaccgga   1020gaagtcccct atcggggcat tgatggcctc gccgtggctt atggggtagc ag#tcaataaa   1080ctcactttgc ccattccatc cacctgccct gagccgtttg ccaagctcat ga#aagaatgc   1140tggcaacaag accctcatat tcgtccatcg tttgccttaa ttctcgaaca gt#tgactgct   1200attgaagggg cagtgatgac tgagatgcct caagaatctt ttcattccat gc#aagatgac   1260tggaaactag aaattcaaca aatgtttgat gagttgagaa caaaggaaaa gg#agctgcga   1320tcccgggaag aggagctgac tcgggcggct ctgcagcaga agtctcagga gg#agctgcta   1380aagcggcgtg agcagcagct ggcagagcgc gagatcgacg tgctggagcg gg#aacttaac   1440attctgatat tccagctaaa ccaggagaag cccaaggtaa agaagaggaa gg#gcaagttt   1500aagagaagtc gtttaaagct caaagatgga catcgaatca gtttaccttc ag#atttccag   1560cacaagataa ccgtgcaggc ctctcccaac ttggacaaac ggcggagcct ga#acagcagc   1620agttccagtc ccccgagcag ccccacaatg atgccccgac tccgagccat ac#agttgact   1680tcagatgaaa gcaataaaac ttggggaagg aacacagtct ttcgacaaga ag#aatttgag   1740gatgtaaaaa ggaattttaa gaaaaaaggt tgtacctggg gaccaaattc ca#ttcaaatg   1800aaagatagaa cagattgcaa agaaaggata agacctctct ccgatggcaa ca#gtccttgg   1860tcaactatct taataaaaaa tcagaaaacc atgcccttgg cttcattgtt tg#tggaccag   1920ccagggtcct gtgaagagcc aaaactttcc cctgatggat tagaacacag aa#aaccaaaa   1980caaataaaat tgcctagtca ggcctacatt gatctacctc ttgggaaaga tg#ctcagaga   2040gagaatcctg cagaagctga aagctgggag gaggcagcct ctgcgaatgc tg#ccacagtc   2100tccattgaga tgactcctac gaatagtctg agtagatccc cccagagaaa ga#aaacggag   2160tcagctctgt atgggtgcac crtccttctg gcatcggtgg ctctgggact gg#acctcaga   2220gakcttcata aagcacaggc tgctgaagaa ccgttgccca aggaagagaa ga#agaaacga   2280gagggaatct tccagcgggc ttccaagtcc cgcagaagyg ccagtcctcc ca#caagcctg   2340ccatccacck gtggggaggc cagcagccca ccctccctgc cactgtcaag tg#ccctgggc   2400atcctctcca caccttcttt ctccacaaag tgcctgctgc agatggacag tg#aagatcca   2460ctggtggaca gtgcacctgt cacttgtgac tctgagatgc tcactccgga tt#tttgtccc   2520actgccccag gaagtggtcg tgagccagcc ctcatgccaa gacttgacac tg#attgtagt   2580gtatcaagaa acttgccgtc ttccttccta cagcagacat gtgggaatgt ac#cttactgt   2640gcttcttcaa aacatagacc rtcacatcac agacggacca tgtctgatgg aa#atccgacc   2700ccaactggtg caactattat ctcagccact ggagcctctg cactgccact ct#gcccctca   2760cctgmtcytc acwgtcatct gccaagggag gtctcaccca agaagcacag ca#ctgtccac   2820atcgtgcctc agcgtcgccc tgcctccctg agaagccgct cagatctgcc tc#aggcttac   2880ccacagacag cagtgtctca gctggcacag actgcctgtg tagtgggtcg cc#caggacca   2940catcccaccc aattcctcgc tgccaaggag agaactaaat cccatgtgcc tt#cattactg   3000gatgctgacg tggaaggtca gagcagggac tacactgtgc cactgtgcag aa#tgaggagc   3060aaaaccagcc ggccatctat atatgaactg gagaaagaat tcctgtctta a #           3111 <210> SEQ ID NO 2 <211> LENGTH: 1036 <212> TYPE: PRT<213> ORGANISM: homo sapiens <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(1036)<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 2Met Ala Leu Arg Gly Ala Ala Gly Ala Thr As #p Thr Pro Val Ser Ser 1               5   #                10   #                15Ala Gly Gly Ala Pro Gly Gly Ser Ala Ser Se #r Ser Ser Thr Ser Ser            20       #            25       #            30Gly Gly Ser Ala Ser Ala Gly Ala Gly Leu Tr #p Ala Ala Leu Tyr Asp        35           #        40           #        45Tyr Glu Ala Arg Gly Glu Asp Glu Leu Ser Le #u Arg Arg Gly Gln Leu    50               #    55               #    60Val Glu Val Leu Ser Gln Asp Ala Ala Val Se #r Gly Asp Glu Gly Trp65                   #70                   #75                   #80Trp Ala Gly Gln Val Gln Arg Arg Leu Gly Il #e Phe Pro Ala Asn Tyr                85   #                90   #                95Val Ala Pro Cys Arg Pro Ala Ala Ser Pro Al #a Pro Pro Pro Ser Arg            100       #           105       #           110Pro Ser Ser Pro Val His Val Ala Phe Glu Ar #g Leu Glu Leu Lys Glu        115           #       120           #       125Leu Ile Gly Ala Gly Gly Phe Gly Gln Val Ty #r Arg Ala Thr Trp Gln    130               #   135               #   140Gly Gln Glu Val Ala Val Lys Ala Ala Arg Gl #n Asp Pro Glu Gln Asp145                 1 #50                 1 #55                 1 #60Ala Ala Ala Ala Ala Glu Ser Val Arg Arg Gl #u Ala Arg Leu Phe Ala                165   #               170   #               175Met Leu Arg His Pro Asn Ile Ile Glu Leu Ar #g Gly Val Cys Leu Gln            180       #           185       #           190Gln Pro His Leu Cys Leu Val Leu Glu Phe Al #a Arg Gly Gly Ala Leu        195           #       200           #       205Asn Arg Ala Leu Ala Ala Ala Asn Ala Ala Pr #o Asp Pro Arg Ala Pro    210               #   215               #   220Gly Pro Arg Arg Ala Arg Arg Ile Pro Pro Hi #s Val Leu Val Asn Trp225                 2 #30                 2 #35                 2 #40Ala Val Gln Ile Ala Arg Gly Met Leu Tyr Le #u His Glu Glu Ala Phe                245   #               250   #               255Val Pro Ile Leu His Arg Asp Leu Lys Ser Se #r Asn Ile Leu Leu Leu            260       #           265       #           270Glu Lys Ile Glu His Asp Asp Ile Cys Asn Ly #s Thr Leu Lys Ile Thr        275           #       280           #       285Asp Phe Gly Leu Ala Arg Glu Trp His Arg Th #r Thr Lys Met Ser Thr    290               #   295               #   300Ala Gly Thr Tyr Ala Trp Met Ala Pro Glu Va #l Ile Lys Ser Ser Leu305                 3 #10                 3 #15                 3 #20Phe Ser Lys Gly Ser Asp Ile Trp Ser Tyr Gl #y Val Leu Leu Trp Glu                325   #               330   #               335Leu Leu Thr Gly Glu Val Pro Tyr Arg Gly Il #e Asp Gly Leu Ala Val            340       #           345       #           350Ala Tyr Gly Val Ala Val Asn Lys Leu Thr Le #u Pro Ile Pro Ser Thr        355           #       360           #       365Cys Pro Glu Pro Phe Ala Lys Leu Met Lys Gl #u Cys Trp Gln Gln Asp    370               #   375               #   380Pro His Ile Arg Pro Ser Phe Ala Leu Ile Le #u Glu Gln Leu Thr Ala385                 3 #90                 3 #95                 4 #00Ile Glu Gly Ala Val Met Thr Glu Met Pro Gl #n Glu Ser Phe His Ser                405   #               410   #               415Met Gln Asp Asp Trp Lys Leu Glu Ile Gln Gl #n Met Phe Asp Glu Leu            420       #           425       #           430Arg Thr Lys Glu Lys Glu Leu Arg Ser Arg Gl #u Glu Glu Leu Thr Arg        435           #       440           #       445Ala Ala Leu Gln Gln Lys Ser Gln Glu Glu Le #u Leu Lys Arg Arg Glu    450               #   455               #   460Gln Gln Leu Ala Glu Arg Glu Ile Asp Val Le #u Glu Arg Glu Leu Asn465                 4 #70                 4 #75                 4 #80Ile Leu Ile Phe Gln Leu Asn Gln Glu Lys Pr #o Lys Val Lys Lys Arg                485   #               490   #               495Lys Gly Lys Phe Lys Arg Ser Arg Leu Lys Le #u Lys Asp Gly His Arg            500       #           505       #           510Ile Ser Leu Pro Ser Asp Phe Gln His Lys Il #e Thr Val Gln Ala Ser        515           #       520           #       525Pro Asn Leu Asp Lys Arg Arg Ser Leu Asn Se #r Ser Ser Ser Ser Pro    530               #   535               #   540Pro Ser Ser Pro Thr Met Met Pro Arg Leu Ar #g Ala Ile Gln Leu Thr545                 5 #50                 5 #55                 5 #60Ser Asp Glu Ser Asn Lys Thr Trp Gly Arg As #n Thr Val Phe Arg Gln                565   #               570   #               575Glu Glu Phe Glu Asp Val Lys Arg Asn Phe Ly #s Lys Lys Gly Cys Thr            580       #           585       #           590Trp Gly Pro Asn Ser Ile Gln Met Lys Asp Ar #g Thr Asp Cys Lys Glu        595           #       600           #       605Arg Ile Arg Pro Leu Ser Asp Gly Asn Ser Pr #o Trp Ser Thr Ile Leu    610               #   615               #   620Ile Lys Asn Gln Lys Thr Met Pro Leu Ala Se #r Leu Phe Val Asp Gln625                 6 #30                 6 #35                 6 #40Pro Gly Ser Cys Glu Glu Pro Lys Leu Ser Pr #o Asp Gly Leu Glu His                645   #               650   #               655Arg Lys Pro Lys Gln Ile Lys Leu Pro Ser Gl #n Ala Tyr Ile Asp Leu            660       #           665       #           670Pro Leu Gly Lys Asp Ala Gln Arg Glu Asn Pr #o Ala Glu Ala Glu Ser        675           #       680           #       685Trp Glu Glu Ala Ala Ser Ala Asn Ala Ala Th #r Val Ser Ile Glu Met    690               #   695               #   700Thr Pro Thr Asn Ser Leu Ser Arg Ser Pro Gl #n Arg Lys Lys Thr Glu705                 7 #10                 7 #15                 7 #20Ser Ala Leu Tyr Gly Cys Thr Val Leu Leu Al #a Ser Val Ala Leu Gly                725   #               730   #               735Leu Asp Leu Arg Glu Leu His Lys Ala Gln Al #a Ala Glu Glu Pro Leu            740       #           745       #           750Pro Lys Glu Glu Lys Lys Lys Arg Glu Gly Il #e Phe Gln Arg Ala Ser        755           #       760           #       765Lys Ser Arg Arg Ser Ala Ser Pro Pro Thr Se #r Leu Pro Ser Thr Gly    770               #   775               #   780Gly Glu Ala Ser Ser Pro Pro Ser Leu Pro Le #u Ser Ser Ala Leu Gly785                 7 #90                 7 #95                 8 #00Ile Leu Ser Thr Pro Ser Phe Ser Thr Lys Cy #s Leu Leu Gln Met Asp                805   #               810   #               815Ser Glu Asp Pro Leu Val Asp Ser Ala Pro Va #l Thr Cys Asp Ser Glu            820       #           825       #           830Met Leu Thr Pro Asp Phe Cys Pro Thr Ala Pr #o Gly Ser Gly Arg Glu        835           #       840           #       845Pro Ala Leu Met Pro Arg Leu Asp Thr Asp Cy #s Ser Val Ser Arg Asn    850               #   855               #   860Leu Pro Ser Ser Phe Leu Gln Gln Thr Cys Gl #y Asn Val Pro Tyr Cys865                 8 #70                 8 #75                 8 #80Ala Ser Ser Lys His Arg Pro Ser His His Ar #g Arg Thr Met Ser Asp                885   #               890   #               895Gly Asn Pro Thr Pro Thr Gly Ala Thr Ile Il #e Ser Ala Thr Gly Ala            900       #           905       #           910Ser Ala Leu Pro Leu Cys Pro Ser Pro Asp Le #u His Xaa His Leu Pro        915           #       920           #       925Arg Glu Val Ser Pro Lys Lys His Ser Thr Va #l His Ile Val Pro Gln    930               #   935               #   940Arg Arg Pro Ala Ser Leu Arg Ser Arg Ser As #p Leu Pro Gln Ala Tyr945                 9 #50                 9 #55                 9 #60Pro Gln Thr Ala Val Ser Gln Leu Ala Gln Th #r Ala Cys Val Val Gly                965   #               970   #               975Arg Pro Gly Pro His Pro Thr Gln Phe Leu Al #a Ala Lys Glu Arg Thr            980       #           985       #           990Lys Ser His Val Pro Ser Leu Leu Asp Ala As #p Val Glu Gly Gln Ser        995           #       1000           #      1005Arg Asp Tyr Thr Val Pro Leu Cys Arg Met Ar #g Ser Lys Thr Ser Arg    1010              #   1015               #  1020Pro Ser Ile Tyr Glu Leu Glu Lys Glu Phe Le #u Ser1025                1030 #                1035 <210> SEQ ID NO 3<211> LENGTH: 3518 <212> TYPE: DNA <213> ORGANISM: homo sapiens<400> SEQUENCE: 3gcagcgccct gggcacgacc atggtgggac gtcgcccgcg gcttcgggga cc#gctgcggc     60agcagaggcg gctggccagg aacgcgggcc gaggctggac cctttgggca gc#tagcccgt    120gatctctgcc gtcaccgatc gcgattccta ccccctcgcc ttcccccggc gc#cgacggcc    180acaccgccgg acgatgcgcg cccgcggccg cccgggaggc tgagcccagc tt#cccgctcc    240gccttccccg cgcagctgcc cccatggctt tgcggggcgc cgcgggagcg ac#cgacaccc    300cggtgtcctc ggccggggga gcccccggcg gctcagcgtc ctcgtcgtcc ac#ctcctcgg    360gcggctcggc ctcggcgggc gcggggctgt gggccgcgct ctatgactac ga#ggctcgcg    420gcgaggacga gctgagcctg cggcgcggcc agctggtgga ggtgctgtcg ca#ggacgccg    480ccgtgtcggg cgacgagggc tggtgggcag gccaggtgca gcggcgcctc gg#catcttcc    540ccgccaacta cgtggctccc tgccgcccgg ccgccagccc cgcgccgccg cc#ctcgcggc    600ccagctcccc ggtacacgtc gccttcgagc ggctggagct gaaggagctc at#cggcgctg    660ggggcttcgg gcaggtgtac cgcgccacct ggcagggcca ggaggtggcc gt#gaaggcgg    720cgcgccagga cccggagcag gacgcggcgg cggctgccga gagcgtgcgg cg#cgaggctc    780ggctcttcgc catgctgcgg caccccaaca tcatcgagct gcgcggcgtg tg#cctgcagc    840agccgcacct ctgcctggtg ctggagttcg cccgcggcgg agcgctcaac cg#agcgctgg    900ccgctgccaa cgccgccccg gacccgcgcg cgcccggccc ccgccgcgcg cg#ccgcatcc    960ctccgcacgt gctggtcaac tgggccgtgc agatagcgcg gggcatgctc ta#cctgcatg   1020aggaggcctt cgtgcccatc ctgcaccggg acctcaagtc cagcaacatt tt#gctacttg   1080agaagataga acatgatgac atctgcaata aaactttgaa gattacagat tt#tgggttgg   1140cgagggaatg gcacaggacc accaaaatga gcacagcagg cacctatgcc tg#gatggccc   1200ccgaagtgat caagtcttcc ttgttttcta agggaagcga catctggagc ta#tggagtgc   1260tgctgtggga actgctcacc ggagaagtcc cctatcgggg cattgatggc ct#cgccgtgg   1320cttatggggt agcagtcaat aaactcactt tgcccattcc atccacctgc cc#tgagccgt   1380ttgccaagct catgaaagaa tgctggcaac aagaccctca tattcgtcca tc#gtttgcct   1440taattctcga acagttgact gctattgaag gggcagtgat gactgagatg cc#tcaagaat   1500cttttcattc catgcaagat gactggaaac tagaaattca acaaatgttt ga#tgagttga   1560gaacaaagga aaaggagctg cgatcccggg aagaggagct gactcgggcg gc#tctgcagc   1620agaagtctca ggaggagctg ctaaagcggc gtgagcagca gctggcagag cg#cgagatcg   1680acgtgctgga gcgggaactt aacattctga tattccagct aaaccaggag aa#gcccaagg   1740taaagaagag gaagggcaag tttaagagaa gtcgtttaaa gctcaaagat gg#acatcgaa   1800tcagtttacc ttcagatttc cagcacaaga taaccgtgca ggcctctccc aa#cttggaca   1860aacggcggag cctgaacagc agcagttcca gtcccccgag cagccccaca at#gatgcccc   1920gactccgagc catacagttg acttcagatg aaagcaataa aacttgggga ag#gaacacag   1980tctttcgaca agaagaattt gaggatgtaa aaaggaattt taagaaaaaa gg#ttgtacct   2040ggggaccaaa ttccattcaa atgaaagata gaacagattg caaagaaagg at#aagacctc   2100tctccgatgg caacagtcct tggtcaacta tcttaataaa aaatcagaaa ac#catgccct   2160tggcttcatt gtttgtggac cagccagggt cctgtgaaga gccaaaactt tc#ccctgatg   2220gattagaaca cagaaaacca aaacaaataa aattgcctag tcaggcctac at#tgatctac   2280ctcttgggaa agatgctcag agagagaatc ctgcagaagc tgaaagctgg ga#ggaggcag   2340cctctgcgaa tgctgccaca gtctccattg agatgactcc tacgaatagt ct#gagtagat   2400ccccccagag aaagaaaacg gagtcagctc tgtatgggtg caccrtcctt ct#ggcatcgg   2460tggctctggg actggacctc agagakcttc ataaagcaca ggctgctgaa ga#accgttgc   2520ccaaggaaga gaagaagaaa cgagagggaa tcttccagcg ggcttccaag tc#ccgcagaa   2580gygccagtcc tcccacaagc ctgccatcca cckgtgggga ggccagcagc cc#accctccc   2640tgccactgtc aagtgccctg ggcatcctct ccacaccttc tttctccaca aa#gtgcctgc   2700tgcagatgga cagtgaagat ccactggtgg acagtgcacc tgtcacttgt ga#ctctgaga   2760tgctcactcc ggatttttgt cccactgccc caggaagtgg tcgtgagcca gc#cctcatgc   2820caagacttga cactgattgt agtgtatcaa gaaacttgcc gtcttccttc ct#acagcaga   2880catgtgggaa tgtaccttac tgtgcttctt caaaacatag accrtcacat ca#cagacgga   2940ccatgtctga tggaaatccg accccaactg gtgcaactat tatctcagcc ac#tggagcct   3000ctgcactgcc actctgcccc tcacctgmtc ytcacwgtca tctgccaagg ga#ggtctcac   3060ccaagaagca cagcactgtc cacatcgtgc ctcagcgtcg ccctgcctcc ct#gagaagcc   3120gctcagatct gcctcaggct tacccacaga cagcagtgtc tcagctggca ca#gactgcct   3180gtgtagtggg tcgcccagga ccacatccca cccaattcct cgctgccaag ga#gagaacta   3240aatcccatgt gccttcatta ctggatgctg acgtggaagg tcagagcagg ga#ctacactg   3300tgccactgtg cagaatgagg agcaaaacca gccggccatc tatatatgaa ct#ggagaaag   3360aattcctgtc ttaaactaag tgccttactg ttgtttaagc atttttttaa gg#tgaacaaa   3420tgaacacaat gtatctacct ttgaactgtt tcatgctgct gtgttttcaa aa#gctgtggc   3480 catgttccta aattagtaag atatatccag cttctcaa      #                   #   3518

What is claimed is:
 1. A substantially isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2.